An example of a so-called "deficient selection marker needed for the growth of the yeast" is the LEU2d gene described by Kingsman c.s. (reference 1) , who described the development of a multicopy integrative vector which was dispersed throughout the genome using the transposable Ty element Ty1-15 (reference 2). The element was engineered to contain two selectable markers, TRP1 (reference 3) and LEU2 from pMA3a, and the PGK expression signals from pMA91 (reference 4) with an IFN-.alpha..sub.2 coding sequence (reference 5). A single copy of the engineered Ty was integrated into the genome using a linear fragment to stimulate recombination across the ends of the element and thereby replacing an endogenous element. Transformants were selected for the TRP1 marker. Few transformants were obtained by selecting for LEU2 as insufficient enzyme was produced by a single copy of this gene. The transformant was then grown in decreasing concentrations of leucine to select for an increase in the copy number of the LEU2 gene, presumably by spread of the Ty element throughout the genome by gene conversion and transposition (reference 6). A strain was constructed which produced 8.times.10.sup.5 molecules of IFN per cell; this being intermediate between yields from single copy ARS/CEN vectors (10.sup.5 molecules/cell) and from multicopy vectors such as pMA91 (6.times.10.sup.6 molecules/cell).
For a practical stable production system with a transformed yeast the use of Ty elements has certain disadvantages.
For example, Ty elements are homologous to retroviral sequences, which are more or less suspect materials for production of a protein suitable for products for human consumption or in the preparation thereof. Thus it is preferable to find solutions whereby these more or less suspect materials are not used.
Another disadvantage is their property of being transposable elements. This has the consequence that an appreciable risk exists that the resulting strain is not genetically stable, because the transposable TY elements integrated in the chromosome of the yeast can transpose and integrate at other sites of the genome which has negative implications for the production process and can give problems in obtaining clearance from responsible companies and the authorities.
In view of their retroviral properties Ty elements may result in virus-like particles. This is highly undesirable for practical production processes, because instability of genetically modified organisms should be avoided.
Ty elements only occur in the yeast Saccharomyces cerevisiae. Therefore it is doubtful, whether they can be used for other yeasts or even moulds. It is unknown whether transposable elements occurring in other organisms can be used in a similar way. But even if they could, they have the same disadvantages as indicated above.
The copy number obtained with Ty integration is about 20-30 with a single maximum of about 40 copies per cell. A higher number of 100-300 copies per cell would be highly advantageous for commercial production systems, as higher copy numbers, in general, will result in higher expression levels.
Therefore a need exists for other systems by which multicopy integration of heterologous genes in fungi such as yeast and moulds can be achieved.